The mortality from lymphoma is high; thus, there is a great need to not only develop new therapeutic agents for this disease but to also further develop agents that have already shown promise in prior investigations. In early clinical trials, gallium nitrate (NSC15200) demonstrated significant clinical activity in lymphoma but its use in lymphoma was never rigorously pursued and information regarding its mechanisms of action has remained largely incomplete. There is now a renewed clinical interest in using gallium for lymphoma and clinical trials are in progress. The long-term objectives of this project are to optimally develop gallium compounds for the treatment of lymphoma by understanding its mechanisms of action. Our preliminary data suggest that the hemochromatosis (HFE) gene influences gallium uptake and cytotoxicity in lymphoblastoid cells and that gallium targets the mitochondria. HFE mutations, C282Y and H63D, occur with high frequency in the population. Specific Aim 1 will further investigate the role of HFE [wild type (wt) and mutant] as modulators of transferrin receptor-targeted gallium uptake and cytotoxicity in lymphoma cells. This Aim will investigate why lymphoblastic cells with the HFE C282Y mutation are more sensitive to gallium than cells with wt HFE. Studies will investigate the effects of HFE on gallium transport, gallium-induced apoptosis, gallium's action on iron homeostasis and ribonucleotide reductase. Specific Aim 2 will examine the effects of gallium on the generation of reactive oxygen species (ROS), mitochondrial function, and apoptosis induction. The effect of HFE mutations on gallium's antineoplastic action will be investigated with immortalized cell lines developed from individuals with wt HFE and HFE mutations. Lymphoma cells transfected with an inducible HFE gene (wt or mutant) will also be utilized. Ga-67 and Fe-59-transferrin will be used for uptake studies. mRNA and protein levels of transferrin receptor, ferritin, and HFE will be measured by Northern blotting, ligand binding, immunoblotting and immunoassays. Iron regulatory protein-RNA binding and ribonucleotide reductase R2 subunit will be studied by bandshift assay and ESR spectroscopy, respectively. ROS in cells will be detected using fluorescent probes and HPLC. Mitochondria-targeted antioxidants will be employed to investigate the source of gallium-induced ROS production. Mitochondrial electron transport enzymes will be assayed by measuring oxygen consumption and ATP production in cells and isolated mitochondria using substrates and inhibitors. Assays will measure aconitase and glucose consumption and caspase activity. Our studies will provide new information regarding: a) the impact of HFE mutations on the response of lymphoma to gallium, and, b) the mechanism of action of gallium at the mitochondrial level.